Effects of carrier solutions on the viability and efficacy of canine adipose-derived mesenchymal stem cells

Background Mesenchymal stem cells (MSCs) have favorable characteristics that render them a potent therapeutic tool. We tested the characteristics of MSCs after temporal storage in various carrier solutions, such as 0.9% saline (saline), 5% dextrose solution (DS), heparin in saline, and Hartmann’s solution, all of which are approved by the U.S. Food and Drug Administration (FDA). Phosphate-buffered saline, which does not have FDA approval, was also used as a carrier solution. We aimed to examine the effects of these solutions on the viability and characteristics of MSCs to evaluate their suitability and efficacy for the storage of canine adipose-derived MSCs (cADMSCs). Results We stored the cADMSCs in the test carrier solutions in a time-dependent manner (1, 6, and 12 h) at 4 °C, and analyzed cell confluency, viability, proliferation, self-renewability, and chondrogenic differentiation. Cell confluency was significantly higher in 5% DS and lower in phosphate-buffered saline at 12 h compared to other solutions. cADMSCs stored in saline for 12 h showed the highest viability rate. However, at 12 h, the proliferation rate of cADMSCs was significantly higher after storage in 5% DS and significantly lower after storage in saline, compared to the other solutions. cADMSCs stored in heparin in saline showed superior chondrogenic capacities at 12 h compared to other carrier solutions. The expression levels of the stemness markers, Nanog and Sox2, as well as those of the MSC surface markers, CD90 and CD105, were also affected over time. Conclusion Our results suggest that MSCs should be stored in saline, 5% DS, heparin in saline, or Hartmann’s solution at 4 °C, all of which have FDA approval (preferable storage conditions: less than 6 h and no longer than 12 h), rather than storing them in phosphate-buffered saline to ensure high viability and efficacy.

Isolation and probiotic potential of lactic acid bacteria from swine feces for feed additive composition

Animal microbiota is becoming an object of interest as a source of beneficial bacteria for commercial use. Moreover, the escalating problem of bacterial resistance to antibiotics is threatening animals and humans; therefore, in the last decade intensive search for alternative antimicrobials has been observed. In this study, lactic acid bacteria (LAB) were isolated from suckling and weaned pigs feces (376) and characterized to determine their functional properties and usability as pigs additives. Selection of the most promising LAB was made after each stage of research. Isolates were tested for their antimicrobial activity (376) and susceptibility to antibiotics (71). Selected LAB isolates (41) were tested for the production of organic acids, enzymatic activity, cell surface hydrophobicity and survival in gastrointestinal tract. Isolates selected for feed additive (5) were identified by MALDI-TOF mass spectrometry and partial sequence analysis of 16S rRNA gene, represented by Lentilactobacillus, Lacticaseibacillus (both previously classified as Lactobacillus) and Pediococcus genus. Feed additive prototype demonstrated high viability after lyophilization and during storage at 4 °C and − 20 °C for 30 days. Finally, feed additive was tested for survival in simulated alimentary tract of pigs, showing viability at the sufficient level to colonize the host. Studies are focused on obtaining beneficial strains of LAB with probiotic properties for pigs feed additive.

IL-10 Rescues CLL Survival through Repolarization of Inflammatory Nurse-like Cells

Tumor-associated macrophages (TAMs) in chronic lymphocytic leukemia (CLL) are also called nurse-like cells (NLC), and confer survival signals through the release of soluble factors and cellular contacts. While in most patient samples the presence of NLC in co-cultures guarantees high viability of leukemic cells in vitro, in some cases this protective effect is absent. These macrophages are characterized by an “M1-like phenotype”. We show here that their reprogramming towards an M2-like phenotype (tumor-supportive) with IL-10 leads to an increase in leukemic cell survival. Inflammatory cytokines, such as TNF, are also able to depolarize M2-type protective NLC (decreasing CLL cell viability), an effect which is countered by IL-10 or blocking antibodies. Interestingly, both IL-10 and TNF are implied in the pathophysiology of CLL and their elevated level is associated with bad prognosis. We propose that the molecular balance between these two cytokines in CLL niches plays an important role in the maintenance of the protective phenotype of NLCs, and therefore in the survival of CLL cells.

Hydrogels with tunable modulus regulate chondrocyte microaggregates growth for cartilage repair

Chondrocyte spheroids in 3D hydrogel are more beneficial to improve their survival and maintain chondrogenic phenotype comparing to dissociated chondrocytes. However, in-situ inducing cell into spheroids rather than encapsulating spheroids in a hydrogel remains a tremendous challenge because of the limitations of biochemical and viscoelastic controllability for hydrogel. Herein, a hydrogel consisting of photo-crosslinkable chitosan methacrylate (CHMA) and semi-interpenetrating polyvinyl alcohol (PVA) is developed as a cell-responsive matrix with controllable viscoelastic properties. The proposed CHMA-PVA precursor preferentially exhibits a weak gel-like state with a storage modulus of 16.9 Pa, loss modulus of 13.0 Pa and yielding stain of 1%, which could allow chondrocyte to vigorously move and assemble but hinder their precipitation before crosslinking. The chondrocytes could form microaggregates within 8 h in vitro and keep high viability. Moreover, subcutaneous implantation experiments demonstrate that the CHMA/PVA hydrogels are biocompatible and degrade within five weeks in vivo. The cell-free hydrogels are further placed in cylindrical cartilage defects in the rabbit femoral condyle and examined 8 weeks postoperatively. Gross, histological and immunohistochemical analyses reveal a significant acceleration for the cartilage regeneration. These findings suggest that this novel cell adhesion-responsive and histo-compatible hydrogel is promising for cartilage regeneration.

Development of Poly-NIsopropylacrylamide Surfaces for the Selection of Swine Sperm

The reproductive efficiency of pig farms is directly correlated with the fertility of the boars. The aim of this work was to develop polymeric materials that can be used as a platform to select a subpopulation of sperm with better cell physiological parameters. Polymeric hydrogels composed of Poly-N-isopropylacrylamide with different positive charges given by copolymerization with (3-acrylamidopropyl) trimethylammonium chloride (APTA, 5-10-15%), were synthesized. Subsequently, the interaction between the sperm cells and the polymeric surfaces was analyzed in TALP medium. Release of the spermatozoa from the polymeric surfaces was induced by changing to Ca2+ free media. Sperm motility, cell viability, plasma membrane and acrosome integrity were evaluated. The results indicated that a higher percentage of swine sperm attached to PNIPAM co-15% APTA hydrogels (62.86±3.33%). Ninety seven percent (97.19±1.45 %) of the sperm released from the PNIPAM co-15%APTA surfaces were viable (p<0.05 vs unbound population and raw semen), with acceptable motility (58.89±1.28%) and with intact plasma and acrosomal membranes (69±1.2% and 98.5±0.65% respectively). These results indicate that hydrogels can be used to select boar sperm with high viability and mobility for use in assisted reproductive techniques.

165 Generating enhanced tumor infiltrating lymphocytes through microfluidic cell squeezing

BackgroundTumor Infiltrating Lymphocyte (TIL) therapies have shown significant solid tumor activity in patients, but current TIL compositions require patient lymphodepletion and high dose IL-2 after cell infusion to support clinical activity. Removing this requirement through ex vivo engineering of the TIL product with mRNA could enhance potency, expand the potential patient population, and potentially allow for repeat dosing and concomitant treatment with checkpoint therapies.MethodsTo transiently overexpress both membrane-bound cytokines and costimulatory molecules, we used microfluidic cell squeezing (Cell Squeeze®) to deliver mRNA directly to the cytosol of expanded tumor reactive CD8 human TILs (AGX-148). After mRNA delivery, the TILs were cultured in media with varying levels of exogenous IL-2 and characterized by flow cytometry.ResultsWe demonstrated that multiple mRNA constructs delivered simultaneously by microfluidic cell squeezing to human TILs are highly expressed (>80% of cells) for multiple days while maintaining high viability (>80%) in vitro. Membrane bound cytokines are able to support cell expansion in the absence of exogenous IL-2 at rates comparable to control cells incubated with a high concentration of IL-2 for up to 3 days. Furthermore, we have identified a membrane-bound cytokine that alters the TIL phenotype as quantified by multiple markers, including increased L-selectin (CD62L), which is an indicator of central memory T cells.ConclusionsThrough microfluidic cell squeeze delivery of mRNAs, we have created enhanced TILs with high levels of membrane-bound cytokines and/or costimulatory molecules in vitro. These cells are able to proliferate without exogenous IL-2 and have an improved phenotype.

Особенности мужского гаметофита томата в условиях вирусного патогенеза и водного дефицита

On the example of F1 hybrid combinations and tomato varieties, the possibility of the assessing method for pollen selection on the responses of male gametophytes under conditions of viral pathogenesis and drought has been shown. It was found the action of factors on the pollen viability and on the rate of pollen tubes growth, leading to the manifestation of differential reactions. The viruses are the main sources of variability of the pollen functional traits, while the effect of water deficit and genotype are considerably weaker. Genotypes that combine the high viability of pollen with the ability to form longer pollen tubes under the complementary action of viruses and water deficit have been identified, suggesting the prospect of these genotypes using in further breeding studies.

Influența factorilor abiotici asupra capacităților germinative a semințelor de fag (Fagus sylvatica L.)

The diversification of beech genotypes on the territory of the Republic of Moldova could be a so-lution to avoid the risks caused by climate change. The objective of this research was to determine the viability of beech seeds collected from four areas of the Ivano-Frankivsk region (Ukraine) and their modi-fication under the influence of various abiotic factors. The analyzed seeds possessed high viability (77.89 – 96.63%), which under the influence of supra optimal temperature +40°C decreased 1.3-1.4 times de-pending on the applied dose. Seeds exposed to heat treatment used reserve substances for radicle growth 1.4 – 1.6 times more than the control.

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication

Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons towards development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here we show a modular hydrogel system for neural 3D cell culture, based on hyaluronan (HA) and poly(ethylene glycol) (PEG), that is cross-linked and functionalized with human recombinant LN 521 using bioorthogonal copper-free click chemistry. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of syringe extruded lt-NES, which can facilitate the development of advanced neuronal tissue and disease models and translation of stem cell-based therapies.

Features of diffusion processes in the preparation of prepregs by the method of layered application of components

The modern production of products made of composite materials based on thermosetting binders is mainly based on the use of pre – impregnated reinforcing technical threads-prepregs. The binder used for such semi-finished products must meet two important technological requirements: have a low reactivity (high viability) when stored in the temperature range from -5 to +25 ° C and the ability to adjust the curing time at the molding temperatures of the product. To eliminate the disadvantages of the traditional method of obtaining polymer composite materials, to improve their strength characteristics and reduce the cost of the resulting reinforced composites, it is proposed to use the method of layered application of components. The essence of the method consists in layer-by-layer impregnation of the fibrous filler with a binder solution, and then a developed curing system consisting of an amine hardener that prevents the interaction of the hardener with the resin under storage conditions and protective polymer emulsions. The binder-filler system is activated only at an elevated temperature under curing conditions. It is established that the optimal parameters for processing by direct pressing of the pre-pegs components obtained by the method of layer deposition are a pressure of 15 MPa and a temperature of160-170 ?С with a pressure exposure of 15 minutes. If you get products by winding, then for such products, heat treatment for 6 hours at a temperature of 120 ?С is optimal. In the conditions of forming products, that is, at an elevated temperature and at an increased pressure, the mutual diffusion of components occurs due to the movement of oncoming flows. Oligomeric molecules from the resin volume diffuse from the inner layer to the outer one, and the components of the curing system meet them from the outer layer to the inner one. The method of layered application of components makes it possible to create a macroheterogenic system of interpenetrating polymer meshes in the contact area of sequentially applied layers. The result of the research is an increase in the shelf life, the viability of prepregs (up to 10 days) and an improvement in the complex of physical and mechanical properties of composites: the destructive stress during static bending increases to 60 %, during dynamic bending (impact) - up to 50 %. The use of carboxymethylcellulose as a protective polymer provides higher indicators of the studied properties than when using butadiene styrene latex as a protective polymer..